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L H Miller

Publications and source records attributed to L H Miller.

At least 19 recordsLinked to original sources
PubMed1996

Trafficking of Plasmodium chabaudi adami-infected erythrocytes within the mouse spleen.

Plasmodium chabaudi adami causes a nonlethal infection in mice. We found that crisis, the time of rapidly dropping parasitemia, was abrogated by splenectomy, indicating the role of spleen in parasite killing. The factors that mediate spleen-dependent immunity are not known. An earlier study in Plasmodium berghei-infected rats showed an association between increased clearance of heat-treated erythrocytes and the onset of crisis [Wyler, D. J., Quinn, T. C. & Chen, L.-T. (1982) J. Clin. Invest. 67, 1400-1404]. To determine the potential effects of different vascular beds in parasite killing, we studied the distribution of parasitized erythrocytes and bacteria in the spleens of P. chabaudi adami-infected mice during precrisis (a period of rising parasitemia) and during crisis. After intravenous injection, bacteria were localized predominantly in the marginal zone. In contrast, parasitized erythrocytes were found in the red pulp. We also found that during precrisis, a time of no immunity, the uptake of radiolabeled infected erythrocytes by the spleen was increased, not decreased. These data imply that no change occurs in the flow of parasitized erythrocytes through the spleen during the transition to an immune state (crisis). Our observations suggest that immune effector mechanisms, not circulatory changes, account for spleen-dependent parasite killing during a P. chabaudi adami infection in mice.

Animals↗
PubMed1995

Isolation of multiple sequences from the Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding proteins.

Open reading frames in the Plasmodium falciparum genome encode domains homologous to the adhesive domains of the P. falciparum EBA-175 erythrocyte-binding protein (eba-175 gene product) and those of the Plasmodium vivax and Plasmodium knowlesi Duffy antigen-binding proteins. These domains are referred to as Duffy binding-like (DBL), after the receptor that determines P. vivax invasion of Duffy blood group-positive human erythrocytes. Using oligonucleotide primers derived from short regions of conserved sequence, we have developed a reverse transcription-PCR method that amplifies sequences encoding the DBL domains of expressed genes. Products of these reverse transcription-PCR amplifications include sequences of single-copy genes (including eba-175) and variably transcribed genes that cross-hybridize to multiple regions of the genome. Restriction patterns of the multicopy genes show a high degree of polymorphism among different parasite lines, whereas single-copy genes are generally conserved. Characterization of the single-copy genes has identified a gene (ebl-1) that is related to eba-175 and is likely to be involved in erythrocyte invasion.

Amino Acid Sequence↗
PubMed1995

Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes.

Plasmodium falciparum expresses on the host erythrocyte surface clonally variant antigens and ligands that mediate adherence to endothelial receptors. Both are central to pathogenesis, since they allow chronicity of infection and lead to concentration of infected erythrocytes in cerebral vessels. Here we show that expression of variant antigenic determinants is correlated with expression of individual members of a large, multigene family named var. Each var gene contains copies of a motif that has been previously shown to bind diverse host receptors; expression of a specific var gene correlated with binding to ICAM-1. Thus, our findings are consistent with the involvement of var genes in antigenic variation and binding to endothelium.

Amino Acid Sequence↗
PubMed1995

A mutant of melanoma growth stimulating activity does not activate neutrophils but blocks erythrocyte invasion by malaria.

Alanine scanning mutagenesis of the charged amino acids of melanoma growth stimulating activity (MGSA) was used to identify specific residues that are involved in binding to the human erythrocyte Duffy antigen/chemokine receptor (DARC) and to the type B interleukin-8 receptor (IL-8RB) on neutrophils. Receptor binding and biological studies with the alanine scan mutants of MGSA demonstrate that MGSA binds to DARC and the IL-8RB through distinct binding regions. One of the MGSA mutants, E6A, binds to human erythrocytes and is able to inhibit malaria invasion as efficiently as wild type MGSA but has a severely reduced ability to bind to or signal through the IL-8RB. Mutant chemokines like E6A could prove to be useful therapeutically for the design of receptor blocking drugs that inhibit erythrocyte invasion by Plasmodium vivax malaria.

Alanine↗
PubMed1995

Plasmodium gallinaceum: a refractory mechanism of ookinete killing in the mosquito, Anopheles gambiae.

We have identified a mechanism for refractoriness to a bird malaria, Plasmodium gallinaceum, in the African vector of human malaria, Anopheles gambiae. Oocysts fail to develop in the refractory mosquitoes as a result of ookinete death which occurs within 27 hr of midgut invasion. Ultrastructural studies showed that parasite death occurs while the ookinete lies free in the midgut epithelial cell cytosol, usually surrounded by an organelle-free zone that consists of finely fibrillar material. The mechanism of parasite killing does not involve a previously described refractory mechanism of parasite encapsulation. We selected genetic lines which are refractory and susceptible to midgut infection. Genetic crossing of the lines suggests that the refractory trait is inherited as a single dominant genetic locus. Other loci probably influence oocyst number in susceptible mosquitoes. Intracellular ookinete killing appears to involve a previously unrecognized host defense mechanism against malaria parasites that involves direct destruction of the invading organism.

Animals↗
PubMed1995

Plasmodium gallinaceum: sporozoite invasion of Aedes aegypti salivary glands is inhibited by anti-gland antibodies and by lectins.

There is evidence which suggests that malaria sporozoites recognize mosquito salivary glands by specific receptor-ligand interactions. We are interested in identifying the putative salivary gland receptor(s) for sporozoite invasion. We used an in vivo bioassay for sporozoite invasion of salivary glands. In this assay, purified sporozoites from mature oocytes of Plasmodium gallinaceum were injected into Aedes aegypti mosquitoes and salivary glands were dissected at different time points after injection. One half of the maximum invasion of salivary glands by sporozoites occurred by 6 hr, and salivary gland sporozoite load did not increase further after 24 hr postinjection. This assay was used to determine the effect of experimental treatments with antibodies and lectins at 24 hr postinjection. We raised a rabbit polyclonal antiserum against female Ae. aegypti salivary glands which recognized tissue-specific determinants in the basal lamina of salivary glands. Purified IgG antibody fraction of the immune serum blocked sporozoite invasion in vivo. We tested a panel of 19 lectins and found 7 which bound to salivary glands. Of these 7, succinylated wheat germ agglutinin and wheat germ agglutinin completely blocked sporozoite invasion; Pisum sativum agglutinin and soybean agglutinin partially blocked; and concanavalin A, Dolichos biflorus agglutinin, and Phaseolus vulgaris erythroagglutinin did not block. Our results suggest that sporozoites interact with glycosylated salivary gland surface molecules which serve as receptors for invasion, and which may be in the salivary gland basal lamina. Because the putative sporozoite receptors contain immunogenic determinants, it is feasible to identify them by an immunological strategy.

Aedes↗
PubMed1995

Plasmodium falciparum protein associated with the invasion junction contains a conserved oxidoreductase domain.

The merozoite cap protein-1 (MCP-1) of Plasmodium falciparum follows the distribution of the moving junction during invasion of erythrocytes. We have cloned the gene encoding this protein from a cDNA library using a monoclonal antibody. The protein lacks a signal sequence and has no predicted transmembrane domains; none of the antisera reacts with the surfaces of intact merozoites, indicating that the cap distribution is submembranous. MCP-1 is divided into three domains. The N-terminal domain includes a 52-amino-acid region that is highly conserved in a large family of bacterial and eukaryotic proteins. Based on the known functions of two proteins of this family and the pattern of amino acid conservation, it is predicted that this domain may possess oxido-reductase activity, since the active cysteine residue of this domain is invariant in all proteins of the family. The other two domains of MCP-1 are not found in any other members of this protein family and may reflect the specific function of MCP-1 in invasion. The middle domain is negatively charged and enriched in glutamate; the C-terminal domain is positively charged and enriched in lysine. By virtue of its positive charge, the C-terminal domain resembles domains in some cytoskeleton-associated proteins and may mediate the interaction of MCP-1 with cytoskeleton in Plasmodium.

Amino Acid Sequence↗
PubMed1995

Cellular mechanisms in the immune response to malaria in Plasmodium vinckei-infected mice.

Infection of mice with the malaria parasite Plasmodium vinckei vinckei is 100% lethal. However, after two infections followed by drug cure, BALB/c mice develop a solid immunity which is antibody independent but mediated by CD4+ T cells. To elucidate the mechanisms of this immunity, spleen cells from immune mice were challenged in vitro with lysates of P. vinckei-infected or uninfected erythrocytes. The parasite antigen induced proliferation of T cells from immune mice but not from nonimmune mice. When gamma interferon production by cells from immune mice was assayed at the single-cell level, 1 to 3 cells per 1,000 cells were found to release this cytokine when exposed to antigen. In contrast, the numbers of interleukin 4 (IL-4)-producing cells from both immune and control mice were < or = 4 per 10(6) cells, regardless of antigen exposure. Investigation in a bioassay showed that P. vinckei antigen induced the release of IL-4 from spleen cells of immune mice but not from those of control mice. Nevertheless, that IL-4 is of minor significance in this system is also suggested by the absence of elevation of immunoglobulin E levels in blood samples from these mice, in contrast to what is seen with P. chabaudi infection, in which IL-4-producing Th2 cells are of major importance for immunity during later phases of infection. Taken together, the present results indicate that immunity to P. vinckei is a Th1 response, with gamma interferon being an important protective factor. Whether or not the Th1 response, through overproduction of tumor necrosis factor alpha, is also responsible for pathology and death in this infection remains to be clarified.

Animals↗
PubMed1995

Immunogenicity and in vivo efficacy of recombinant Plasmodium falciparum merozoite surface protein-1 in Aotus monkeys.

BACKGROUND: The carboxy-terminus of the merozoite surface protein-1 (MSP1) of Plasmodium falciparum has been implicated as a target of protective immunity. MATERIALS AND METHODS: Two recombinant proteins from the carboxy-terminus of MSP1, the 42 kD fused to GST (bMSP1(42)) and the 19 kD (yMSP1(19)), were expressed in Escherichia coli and secreted from Saccharomyces cerevisiae, respectively. To determine if vaccination with these recombinant proteins induces protective immunity, we conducted a randomized, blinded vaccine trial in two species of Aotus monkeys, A. nancymai and A. vociferans. After three injections using Freund's adjuvant, the monkeys were challenged with the virulent Vietnam Oak Knoll (FVO) strain of P. falciparum. RESULTS: All three control monkeys required treatment by Day 19. Two of three monkeys vaccinated with bMSP1(42) required treatment by Day 17, whereas the third monkey controlled parasitemia for 28 days before requiring treatment. In contrast, both of the A. nancymai vaccinated with yMSP1(19) self-resolved an otherwise lethal infection. One of the two yMSP1(19)-vaccinated A. vociferans had a prolonged prepatent period of > 28 days before requiring treatment. No evidence of mutations were evident in the parasites recovered after the prolonged prepatent period. Sera from the two A. nancymai that self-cured had no detectable effect on in vitro invasion. CONCLUSIONS: Vaccination of A. nancymai with yMSP1(19) induced protective immune responses. The course of recrudescing parasitemias in protected monkeys suggested that immunity is not mediated by antibodies that block invasion. Our data indicate that vaccine trials with the highly adapted FVO strain of P. falciparum can be tested in A. nancymai and that MSP1(19) is a promising anti-blood-stage vaccine for human trials.

Amino Acid Sequence↗
PubMed1994

Analysis of human T cell clones specific for conserved peptide sequences within malaria proteins. Paucity of clones responsive to intact parasites.

T cells are thought to be of central importance in malaria immunity. Peptides copying malaria protein sequences often stimulate human CD4+ T cells and it was thought that they represented T cell epitopes present in the parasite and may thus have particular relevance to malaria vaccine development. To verify whether synthetic peptides representing highly conserved regions of parasite Ags may contribute to a malaria vaccine, we searched the data bank for conserved regions of Plasmodium falciparum malaria proteins that were not homologous to known self (human) proteins. We synthesized 24 such peptides representing 11 of the cloned and sequenced malaria asexual stage Ags, which were predicted by algorithms to represent T cell epitopes, and 6 peptides not predicted to be T cell epitopes and used these to generate T cell clones from individuals with an extensive previous history of malaria exposure. The T cell clones responded vigorously to many peptides but only a single clone, specific for a peptide within merozoite surface protein-1, 20-39, VTHESYQELVKKLEALEDAV, and not previously defined to be a T cell epitope responded to malaria parasites by proliferation and secretion of IFN-gamma. This epitope was not revealed by studying parasite-induced T cell lines and is thus subdominant. The clone was able to significantly inhibit parasite growth in vitro. The final step in the inhibition of parasite growth appears to be nonspecific because other activated clones (not specific for malaria sequences) can inhibit parasite growth. Our data suggest that few conserved peptides within malaria parasites can be processed from the intact parasite. However, such peptides that can be processed from malaria parasites may be expected to stimulate parasite-specific T cells that could inhibit parasite growth and as such may be lead candidates for a vaccine aimed at inducing cellular immunity to malaria.

Adult↗
PubMed1994

Identification of the erythrocyte binding domains of Plasmodium vivax and Plasmodium knowlesi proteins involved in erythrocyte invasion.

Plasmodium vivax and the related monkey malaria, P. knowlesi, require interaction with the Duffy blood group antigen, a receptor for a family of chemokines that includes interleukin 8, to invade human erythrocytes. One P. vivax and three P. knowlesi proteins that serve as erythrocyte binding ligands in such interactions share sequence homology. Expression of different regions of the P. vivax protein in COS7 cells identified a cysteine-rich domain that bound Duffy blood group-positive but not Duffy blood group-negative human erythrocytes. The homologous domain of the P. knowlesi proteins also bound erythrocytes, but had different specificities. The P. vivax and P. knowlesi binding domains lie in one of two regions of homology with the P. falciparum sialic acid binding protein, another erythrocyte binding ligand, indicating conservation of the domain for erythrocyte binding in evolutionarily distant malaria species. The binding domains of these malaria ligands represent potential vaccine candidates and targets for receptor-blockade therapy.

Animals↗
PubMed1994

Malaria pathogenesis.

Malaria is a disease caused by repeated cycles of growth of the parasite Plasmodium in the erythrocyte. Various cellular and molecular strategies allow the parasite to evade the human immune response for many cycles of parasite multiplication. Under certain circumstances Plasmodium infection causes severe anemia or cerebral malaria; the expression of disease is influenced by both parasite and host factors, as exemplified by the exacerbation of disease during pregnancy. This article provides an overview of malaria pathogenesis, synthesizing the recent field, laboratory, and epidemiological data that will lead to the development of strategies to reduce mortality and morbidity.

Anemia↗
PubMed1994

Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum.

A 175-kilodalton erythrocyte binding protein, EBA-175, of the parasite Plasmodium falciparum mediates the invasion of erythrocytes. The erythrocyte receptor for EBA-175 is dependent on sialic acid. The domain of EBA-175 that binds erythrocytes was identified as region II with the use of truncated portions of EBA-175 expressed on COS cells. Region II, which contains a cysteine-rich motif, and native EBA-175 bind specifically to glycophorin A, but not to glycophorin B, on the erythrocyte membrane. Erythrocyte recognition of EBA-175 requires both sialic acid and the peptide backbone of glycophorin A. The identification of both the receptor and ligand domains may suggest rational designs for receptor blockade and vaccines.

Animals↗
PubMed1994

Impact of malaria on genetic polymorphism and genetic diseases in Africans and African Americans.

The high mortality from malaria in sub-Sahara Africa selected multiple genes that give the population a selective advantage. Identification of the genetic basis for resistance may suggest unusual approaches to development of malarial vaccines and antimalarial drugs. Some of these genes may be deleterious, although of selective advantage within the African setting, and need to be identified for counseling for disease prevention.

Africa South of the Sahara↗
PubMed1994

Plasmodium gallinaceum: differential lysis of two developmental stages of malaria sporozoites by the alternative pathway of complement.

During sporogonic development of Plasmodium gallinaceum in the mosquito vector, two developmentally distinct sporozoite stages can be isolated. Sporozoites obtained from oocysts in abdomens of mosquitoes 10 days after an infective blood meal are poorly infectious to the vertebrate host (chicken); days later, sporozoites isolated from mosquito salivary glands are highly infectious. In a first step toward understanding the physiologic basis of this developmentally regulated infectivity to the vertebrate host, we determined the relative resistance of the two sporozoite stages to lysis by the complement system of the vertebrate host. Whereas 86% of oocyst sporozoites were lysed when incubated in fresh chicken serum in vitro, only 24% of salivary gland sporozoites were lysed under identical incubation conditions. Preincubation of oocyst sporozoites in a homogenate of female Aedes aegypti salivary glands did not diminish their susceptibility to lysis by serum, indicating that lysis was mediated by the interaction of serum factors with parasite-specific molecules. The lytic activity of fresh chicken serum was abrogated by incubating for 45 min at 56 degrees C or chelating with EDTA. Fresh chicken serum specifically depleted of Ca2+, by chelating with EGTA in the presence of Mg2+, retained its ability to lyse oocyst sporozoites. The lysis of salivary gland sporozoites mediated by chicken antisporozoite serum is abrogated by EGTA, indicating that the antibody-dependent complement pathway in chicken serum is blocked by EGTA. Because oocyst sporozoite lysis by serum was heat sensitive and Mg2+ dependent, but Ca2+ independent, we conclude that lysis was mediated by the alternative pathway of complement.(ABSTRACT TRUNCATED AT 250 WORDS)

Aedes↗
PubMed1994

Staurosporine inhibits invasion of erythrocytes by malarial merozoites.

Staurosporine, a protein kinase inhibitor, inhibits the invasion of rhesus by Plasmodium knowlesi merozoites with an IC50 of 250 nM. The drug exerts its effects primarily on the merozoite, with little or no effect on the erythrocyte. Okadaic acid, an inhibitor of protein phosphatases, can partially abrogate the inhibitory effects of staurosporine. Staurosporine arrests invasion at a step which is ultrastructurally similar to the arrest caused by cytochalasins B and D: the merozoite attaches, apically reorients, and forms a junction with the erythrocyte, but it does not internalize. These results suggest that protein phosphorylation within the merozoite plays an important role in the internalization step of invasion.

Alkaloids↗